Bottom Line:
Ribonucleic acid (RNA) modifications play an important role in the regulation of gene expression and the development of RNA-based therapeutics, but their identification, localization and relative quantitation by conventional biochemical methods can be quite challenging.We found that CAD of RNA is a stepwise reaction that is facilitated by, but does not require, the presence of positive charge.Preferred backbone cleavage next to adenosine and guanosine in CAD of (M+nH)(n+) and (M-nH)(n-) ions, respectively, is based on hydrogen bonding between nucleobase and phosphodiester moieties.

Mentions:
Previous studies have shown that CAD of lowly charged (M−nH)n− ions of RNA (∼0.2 charges/nt, 8–76 nt) produces primarily (up to 98%) (23,24) c- and y-type fragments from phosphodiester bond cleavage (Scheme 1) (23,24,27,39,47), and that the number of a- and w-type fragments from alternative C3′–O bond cleavage increases with increasing (M−nH)n− ion net charge (24,39). In this study, we find that CAD of (M+nH)n+ ions of RNA also yields predominantly c and y, but virtually no a and w fragments, even at >0.2 charges/nt, as illustrated for RNA 2 (13 nt) with n = 4 (0.31 charges/nt) in Figure 2. Under conditions that gave ∼50% c and y fragments, using 44 and 72 eV (Figures 2 and 3A) for CAD of (M+4H)4+ and (M−4H)4− ions, respectively, the branching ratio between c, y and a, w fragments was ∼3:1 for negatively and ∼30:1 for positively charged RNA 2; similar behavior was observed for all other RNAs studied. Note that at these energies, 44 and 72 eV, the RNA ions dissociated to a similar extent, ∼75% for all (M+4H)4+ and ∼80% for all (M−4H)4− ions, respectively, such that the 10-fold difference in branching ratio is primarily a result of ion polarity rather than CAD energy.

Mentions:
Previous studies have shown that CAD of lowly charged (M−nH)n− ions of RNA (∼0.2 charges/nt, 8–76 nt) produces primarily (up to 98%) (23,24) c- and y-type fragments from phosphodiester bond cleavage (Scheme 1) (23,24,27,39,47), and that the number of a- and w-type fragments from alternative C3′–O bond cleavage increases with increasing (M−nH)n− ion net charge (24,39). In this study, we find that CAD of (M+nH)n+ ions of RNA also yields predominantly c and y, but virtually no a and w fragments, even at >0.2 charges/nt, as illustrated for RNA 2 (13 nt) with n = 4 (0.31 charges/nt) in Figure 2. Under conditions that gave ∼50% c and y fragments, using 44 and 72 eV (Figures 2 and 3A) for CAD of (M+4H)4+ and (M−4H)4− ions, respectively, the branching ratio between c, y and a, w fragments was ∼3:1 for negatively and ∼30:1 for positively charged RNA 2; similar behavior was observed for all other RNAs studied. Note that at these energies, 44 and 72 eV, the RNA ions dissociated to a similar extent, ∼75% for all (M+4H)4+ and ∼80% for all (M−4H)4− ions, respectively, such that the 10-fold difference in branching ratio is primarily a result of ion polarity rather than CAD energy.

Bottom Line:
Ribonucleic acid (RNA) modifications play an important role in the regulation of gene expression and the development of RNA-based therapeutics, but their identification, localization and relative quantitation by conventional biochemical methods can be quite challenging.We found that CAD of RNA is a stepwise reaction that is facilitated by, but does not require, the presence of positive charge.Preferred backbone cleavage next to adenosine and guanosine in CAD of (M+nH)(n+) and (M-nH)(n-) ions, respectively, is based on hydrogen bonding between nucleobase and phosphodiester moieties.